Screen For Interacting Proteins Of Botrytis Cinerea-related Grape VaJAZ9 And Transformation Study Of Grape Thompson Seedless

Grape is one of the most economically important fruits around the world.European grape(Vitis vinifera L.)cultivated widely nowadays is inclined to be infected by various disease,resulting in poor productivity and fruit quality.With the rapid development of protected cultivation around our country,grape production is currently threatened by gray mold disease.Previous studies showed that jasmonates(JAs)is mainly responsible for regulating the biological process in response to necrotrophic pathogens.As a key component in jasmonate signaling pathway,JAZ proteins are involved in perceiving jasmonate signal and associate with a number of transcription factors to regulate corresponding JAs-mediated reaction.However,at present,the research with regard to JAZ protein mainly focuses on the model plant Arabidopsis,the function of JAZ proteins in grape remain poorly understood.In this study,we characterized VaJAZ9 gene as well as its promoter from Chinese wild grape(Vitis amurensis),and revealed its expression pattern and protein localization.Meanwhile,the screen for interacting proteins of VaJAZ9 using yeast two hybrid system was also carried out,and the potential candidates were confirmed in yeast.In addition,the VaJAZ9 over-expressing as well as knock-out vector were constructed and transformed into Thompson Seedless,and transgenic grape seedlings were generated.The main results are as followed:1.The cloned VaJAZ9 cDNA was 807 bp long and would encode a putative protein of 268 amino acids.This protein contained a TIFY domain in the middle and and aJas domain near its C terminus.VaJAZ9 shared close relationship with At JAZ1/2 in Arabidopsis and Sl JAZ2 in tomato,while was phylogenetically distant from JAZ family in rice.2.It was found that VaJAZ9 was relatively strongly expressed in flowers,roots and leaves,and weakly expressed in stems and fruits.Beside that,VaJAZ9 was significantly up-regulated upon Me JA treatment and inoculation of Botrytis cinerea.The VaJAZ9 promoter region was isolated and analysed.It contained several cis elements related to stress response including CGTCA-motif,TC-rich repeats,W-box and G-box.Subcellular localization assay indicated that VaJAZ9 was targeted to the nucleus.3.The interacting proteins of VaJAZ9 were screened by yeast two hybrid system.Fristly,bait vector was established and transformed into yeast competent cells.Autoactivation assay suggested that VaJAZ9 did not exhibit any transcriptional activity.The results of interaction with library and subsequent confirmation showed that VaJAZ9 was associated with Va RGA in yeast.4.Embryogenic callus of Thompson Seedless was induced both from flower buds and somatic embryo.VaJAZ9 coding sequence was isolated and fused to over-expression vector.Meanwhile,with application of CRISPR/Cas9 system,two targets sites were designed at the second exon of VaJAZ9 and corresponding gene edit vector were constructed.Both vectors were introduced into proembryonal masses using Agrobacterium-mediated genetic transformation.After antibiotic screening,seedling germination and other related processes,a certain number of transgenic grape plants were finally obtained.